Naltrexone modulates dopamine release following chronic, but not acute amphetamine administration: a translational study.

The opioid antagonist naltrexone has been shown to attenuate the subjective effects of amphetamine. However, the mechanisms behind this modulatory effect are currently unknown. We hypothesized that naltrexone would diminish the striatal dopamine release induced by amphetamine, which is considered an important mechanism behind many of its stimulant properties. We used positron emission tomography and the dopamine D2-receptor radioligand [11C]raclopride in healthy subjects to study the dopaminergic effects of an amphetamine injection after pretreatment with naltrexone or placebo. In a rat model, we used microdialysis to study the modulatory effects of naltrexone on dopamine levels after acute and chronic amphetamine exposure. In healthy humans, naltrexone attenuated the subjective effects of amphetamine, confirming our previous results. Amphetamine produced a significant reduction in striatal radioligand binding, indicating increased levels of endogenous dopamine. However, there was no statistically significant effect of naltrexone on dopamine release. The same pattern was observed in rats, where an acute injection of amphetamine caused a significant rise in striatal dopamine levels, with no effect of naltrexone pretreatment. However, in a chronic model, naltrexone significantly attenuated the dopamine release caused by reinstatement of amphetamine. Collectively, these data suggest that the opioid system becomes engaged during the more chronic phase of drug use, evidenced by the modulatory effect of naltrexone on dopamine release following chronic amphetamine administration. The importance of opioid-dopamine interactions in the reinforcing and addictive effects of amphetamine is highlighted by the present findings and may help to facilitate medication development in the field of stimulant dependence.

Effect of growth hormone on the translocation of GLUT4 and its relation to insulin-like and anti-insulin action.

To elucidate the effect of growth hormone (GH) on the insulin signal transduction pathway leading to the translocation of glucose transporter-4 (GLUT4), we constructed Chinese hamster ovary cells that overexpressed GH receptor and GLUT4. Treatment with GH triggered GLUT4 translocation, and this translocation was completely inhibited by wortmannin. GH-induced GLUT4 translocation reached a maximum level after 30 min, and then gradually decreased and returned to the basal level after 2 h. Tyrosine phosphorylation of JAK2 also became maximal after 30 min and then gradually decreased. In contrast, GLUT4 translocation remained unchanged for 2 h after insulin treatment, and tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) also remained constant for up to 2 h. Chronic GH treatment had almost no effect on insulin-stimulated Akt kinase activation and GLUT4 translocation. These results suggest that GH and insulin translocate GLUT4 in a similar manner, at least in part, and the difference in translocation depends on the difference in the tyrosine phosphorylation of JAK2 and IRS-1. The anti-insulin action of GH after chronic GH treatment does not appear to be mainly due to the inhibition of GLUT4 translocation.

Vitamin D-dependent rickets type II: regulation of human osteocalcin gene expression in cells with defective vitamin D receptors by 1,25-dihydroxyvitamin D-3, retinoic acid, and triiodothyronine.

The vitamin D receptor (VDR) is a nuclear transcription factor which binds to the vitamin D response element (VDRE) of the human osteocalcin gene and regulates its expression. Humans with VDR gene mutations, ever among those with the same point mutation in their VDR gene, demonstrate clinical heterogeneity. In addition, in some patients with these mutations, rickets has not recurred following cessation of therapy during follow-up ranging from 6 to 24 years. While important, it is likely that the VDR protein is not the sole factor in the development of rickets. To try to understand these clinical findings, the complex formed between the VDRE and one or more proteins in the nuclear extracts of cultured skin fibroblasts treated with 1,25-dihydroxyvitamin D-3 (1,25(OH)2D3), retinoic acid (RA), and/or triiodothyronine (T3) was investigated since such complexes are likely to precede the transcription of the VDR gene. Complex formation in the control cells with an intact VDR was increased by treatment with either 0.1 nM, 1 nM, 10 nM 1,25(OH)2D3, 100 nM RA, or 100 nM T3; however, combinations of these compounds did not produce an additive effect. In cells of affected patients, 1,25(OH)2D3, RA, or T3 increased complex formation, while no combination had an additive effect. These results indicate that 1,25(OH)2D3, RA, and T3 play a role in the regulation of bone remodeling through modulating the formation of protein complexes on the VDRE. Therefore, the clinical observations in patients with a VDR mutation might be explained at least in part by the overlapping control of osteocalcin expression by 1,25(OH)2D3, RA and T3.

25-Hydroxyvitamin D-24-hydroxylase in phytohemagglutinin-stimulated lymphocytes: intermediate bioresponse to 1,25-dihydroxyvitamin D3 of cells from parents of patients with vitamin D-dependent rickets type II.

A method for assay of 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase) activity in phytohemagglutinin (PHA)-stimulated lymphocytes was applied to determine whether vitamin D-dependent rickets type II (VDDR II) is hereditary. In normal lymphocytes incubated with PHA for 3 days, maximal and half-maximal responses of 24-hydroxylase were observed after exposure to 10(-8) mol/L and (1.3 +/- 0.4) x 10(-9) mol/L 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], respectively. These responses were similar to those of cultured skin fibroblasts. In contrast, after exposure to 10(-8), 10(-7), and 10(-6) mol/L 1,25-(OH)2D3, no 24-hydroxylase activity was detected in cells from patients with VDDR II, and intermediate activity was observed in cells from their parents. These findings indicated the presence of an intracellular receptor-effector system for 1,25-(OH)2D3 in peripheral lymphocytes. Heterozygotes of VDDR II could be identified, and autosomal recessive inheritance of the disease was demonstrated. Detection of heterozygotes of this disease was not possible by assay of inhibition of thymidine incorporation, another marker of the function of 1,25-(OH)2D3 in PHA-stimulated lymphocytes. Therefore, assay of 24-hydroxylase induction reflected the receptor status more closely than assay of inhibition of DNA biosynthesis. The assay of 24-hydroxylase activity in PHA-stimulated lymphocytes described here will be useful for diagnosis of VDDR II and study of families of patients with this disease.

Studies on antianaphylactic agents. 4. Synthesis and structure-activity relationships of 3-(4-oxo-4H-1-benzopyran-3)acrylic acids, a new series of antiallergic substances, and some related compounds.

The syntheses of trans-3-(4-oxo-4H-1-benzypyran-3)acrylic acid and a number of analogs shown to be highly active in antiallergic bioassays are described. These compounds are of possible value in the treatment of asthma. The structural requirements for biological activity are discussed with reference to the type of the substituents on the chromone ring or positions of linkage of the acrylic acid on the pyrone ring.

Studies on antianaphylactic agents. 5. Synthesis of 3-(1H-tetrazol-5-yl)chromones, a new series of antiallergic substances.

A number of 3-(1H-tetrazol-5-yl)chromones were synthesized and found to have antiallergic activity in the rat passive cutaneous anaphylaxis (PCA) test. These compounds are active when administered orally in rats and of possible value for the treatment of asthma.

Studies on antianaphylactic agents. 6. Synthesis of some metabolites of 6-ethyl-3-(1H-tetrazol-5-yl)chromone and their analogues.

The metabolites of 6-ethyl-3-(1H-tetrazol-5-yl)chromone (AA-344) (1), an orally effective antiallergic agent, and their analogues were synthesized to confirm the proposed structures and to determine their activity in the rat passive cutaneous anaphylaxis (PCA) test. A glucuronic acid metabolite (6) was assigned the structure 24b, 1-deoxy-1-[5-(6-ethylchromon-3-yl)tetrazol-1-yl]-beta-D-glucopyranuronate, by the comparison of 13C NMR, mass spectra, and TLC of isomeric compounds. In 13C NMR spectra, the shift difference of the tetrazole ring carbons between a pair of isomers was more remarkable than that of the glycosidic carbons. Therefore, the former is a useful criterion for distinguishing between such isomers. Some of the metabolities and analogues were active when administered intravenously, and two metabolites (2 and 3) were also effective upon oral administration.

1 alpha-hydroxyvitamin D3 treatment of three patients with 1,25-dihydroxyvitamin D-receptor-defect rickets and alopecia.

Three patients with clinically different severities of vitamin D-dependent rickets, type II, with alopecia, which is 1,25-dihydroxyvitamin D-receptor-defect rickets and is particularly resistant to treatment with calciferol analogues, were treated with large doses of 1 alpha-hydroxyvitamin D3 (1 alpha-(OH)D3) and 2 g of calcium lactate. Except for the alopecia, all of the abnormalities of patients 1 and 2 were reversed by treatment with 3 micrograms/kg/d of 1 alpha-(OH)D3, and those of patient 3, who had the severest manifestations, were reversed by treatment with 6 micrograms/kg/d. The serum 24,25-dihydroxyvitamin D concentrations of the three patients were low before treatment and those of patients 1 and 2 increased during treatment. These findings suggest that in patients 1 and 2, 25-hydroxyvitamin D-24-hydroxylase was stimulated via a 1,25-dihydroxyvitamin D-receptor-mediated system by treatment with 1 alpha-(OH)D3.

Concomitant administration of sodium dichloroacetate and thiamine in west syndrome caused by thiamine-responsive pyruvate dehydrogenase complex deficiency.

We treated a female patient with West syndrome caused by thiamine-responsive pyruvate dehydrogenase complex (PDHC) deficiency. Infantile spasms occurred in association with elevated blood and CSF lactate concentrations; these symptoms disappeared when lactate concentrations had been lowered by treatment with concomitant sodium dichloroacetate (DCA) and high dose thiamine. Sequencing the patient's PDHC E(1)alpha subunit revealed a substitution of serine for glycine at position 89 in exon 3 (G89S). This mutation must be a de novo mutation because it was not found in either parents' genome DNA. To our knowledge, five previously described patients with PDHC deficiency have displayed the West syndrome. All six known patients, including our own, were female, even though an approximately equal number of males and females have been identified with PDHC deficiency and overall West syndrome occurs somewhat more frequently in males. These results indicated that West syndrome occurred more frequently in female patients with PDHC deficiency. It is suggested that lactate concentration should be measured in patients with West syndrome for potential PDHC deficiency, especially in females.

Rapid diagnosis of vitamin D-dependent rickets type II by use of phytohemagglutinin-stimulated lymphocytes.

The interactions of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] with phytohemagglutinin (PHA)-stimulated lymphocytes from normal subjects and three patients with vitamin D-dependent rickets (DDR) type II were investigated. Impaired nuclear uptake and normal cytosol binding of [3H]1,25-(OH)2D3 were observed with PHA-stimulated lymphocytes of these patients as with their cultured skin fibroblasts. Furthermore, the incorporation of [14C]thymidine into PHA-stimulated lymphocytes of the patients was not reduced by 1,25-(OH)2D3, which is known to inhibit proliferation of various cells. These findings suggest that 1,25-(OH)2D3 receptors are reduced or absent in patients with DDR type II. Thus, the capacities of cytosol binding and nuclear uptake of 1,25-(OH)2D3 in PHA-stimulated lymphocytes seem to reflect those of endo-organs such as the intestine and bone. These findings show that a test of the effect of 1,25-(OH)2D3 on thymidine incorporation into PHA-stimulated lymphocytes is useful for rapid diagnosis of DDR type II.

Decrease of pyruvate dehydrogenase phosphatase activity in patients with congenital lactic acidemia.

We developed an assay method for pyruvate dehydrogenase phosphatase activity using [1-14C]pyruvate and measured pyruvate dehydrogenase phosphatase activity in cultured skin fibroblasts from three patients with congenital lactic acidemia due to a defect in activation of the pyruvate dehydrogenase complex. The enzyme activity of their fibroblasts was significantly reduced to 50.7%, 64.6% and 63.1% of that of control fibroblasts. These observations suggest that the defect in activation of the pyruvate dehydrogenase complex in these patients might be due to a reduction in pyruvate dehydrogenase phosphatase activity.

Screening for disorders of pyruvate metabolism by measuring the ratio of the rates of lactate production and pyruvate decarboxylation in cultured skin fibroblasts.

We assayed the rates of lactate production from [1-14C]pyruvate and decarboxylation of [1-14C]pyruvate in cultured skin fibroblasts from 8 patients with disorders of pyruvate metabolism and 16 control subjects. The disorders of pyruvate metabolism could be more readily detected by measuring the ratio between the rates of lactate production and pyruvate decarboxylation by cultured skin fibroblasts than by measuring either the rate in isolation.

Hemifacial spasm caused by a spontaneous dissecting aneurysm of the vertebral artery. Case report.

The authors describe the first reported case of dissecting aneurysm presenting with hemifacial spasm. The patient was a 58-year-old woman with left hemifacial spasm of 2 years' duration. Cranial nerve examination was otherwise normal and no other clinical symptoms were observed. Vertebral angiography revealed a fusiform enlargement of the left vertebral artery and contrast medium remaining in the intramural false lumen in the venous phase. Microvascular decompression of the facial nerve with wrapping of the aneurysm resulted in complete relief of the hemifacial spasm.

Molecular genetic analysis of pyridoxine-nonresponsive homocystinuric siblings with different blood methionine levels during the neonatal period.

Two mutations in the cystathionine beta-synthase (CBS) gene were found in two Japanese siblings with pyridoxine non-responsive homocystinuria who had different methionine levels in their blood during the neonatal period. Both patients were compound heterozygotes of two mutant alleles: one had an A-to-G transition at nucleotide 194 (A194 G) that caused a histidine-to-arginine substitution at position 65 of the protein (H65R), while the other had a G-to-A transition at nucleotide 346 (G346A) which resulted in a glycine-to-arginine substitution at position 116 of the protein (G116R). The two mutant proteins were separately expressed in Escherichia coli, and they completely lacked catalytic activity. Despite their identical genotypes and almost equal protein intake, these siblings showed different levels of blood methionine during the neonatal period, suggesting that the level of methionine in blood is determined not only by the defect in the CBS gene and protein intake, but also by the activity of other enzymes involved in methionine and homocysteine metabolism, especially during the neonatal period. Therefore, high-risk newborns who have siblings with homocystinuria, even if the level of methionine in their blood is normal in a neonatal mass screening, should be followed up and diagnosed by an assay of enzyme activity or a gene analysis so that treatment can be begun as soon as possible to prevent the development of clinical symptoms. In addition, a new, more sensitive method for the mass screening of CBS deficiency in neonates should be developed.

Effect of sodium dichloroacetate on human pyruvate metabolism.

Sodium dichloroacetate (DCA) was administered orally at doses of 12.5 to 50 mg/kg body weight twice or three times per day to a patient with mitochondrial encephalomyopathy associated with congenital lactic acidemia. During therapy, the rates of decarboxylation of (1-14C) pyruvate and (3-14C) pyruvate, which represent the activity of the pyruvate dehydrogenase (PDH) complex and the function of the TCA cycle, respectively, were markedly increased in the platelets and increases in the lactate levels in the blood and urine during exercise were markedly reduced. These results suggest that oral administration of DCA causes significant increases in the activities of the PDH complex and TCA cycle not only in the platelets but also in various tissues of humans, which is important as a pathway for production of energy, resulting in decreases in the lactate and pyruvate levels in the blood and cerebrospinal fluid.

Reproducibility of [11 C]FLB 457 binding in extrastriatal regions.

Extrastriatal D2 dopamine receptors represent an important target of research into the pathophysiology and pharmacotherapy of psychiatric disorders. The high affinity radioligand [11C]FLB 457 makes possible the measurement of low concentrations of D2 receptors in extrastriatal regions using positron emission tomography (PET). The aim of this study was to assess the test/retest variability and reliability of [11C]FLB 457 binding using a reference tissue model. Eight healthy male subjects (aged 20-33 years) underwent two [11C]FLB 457 PET examinations. Radioactivity in the cerebellum was used as the reference. The binding potentials (BPs) for five cortical regions of interest (ROIs) were calculated using the reference tissue model. The BP was also calculated for each pixel in the form of parametric images. Reproducibility was assessed both for the ROI method and for the parametric images. The test/retest reproducibility for [11C]FLB 457 binding was good, with a mean variability ranging from 4.5% for the thalamus to 15.5% for the hippocampus. The parametric images also demonstrated good reproducibility. These results support the suitability of using [11C]FLB 457 for the quantitative evaluation of extrastriatal D2 receptors and for protocols requiring repeated measurements in the same individual.

[A procedure for electroretinogram (ERG) recording in mice--effect of monoiodoacetic acid on the ERG in pigmented mice].

A procedure for recording the electroretinogram (ERG) in pigmented mice, C57BL, based on the ERG recording technique reported previously in albino mice, ICR, and giving careful consideration to the influence of anesthetics and dark-adaptation, was developed in order to examine retinal toxicity. Pigmented mice were given a single i.v. injection of monoiodoacetic acid (IAA), a known retinotoxic compound, via a tail vein at a dose of 30, 45 or 60 mg/kg, and the ERG was recorded periodically over the next 14 days. In addition, the retinas were examined histopathologically on day 15. The results were as follows. 1. The oscillatory potentials were not distinct in the ERGs from mice anesthetized with pentobarbital as compared to the ERGs from non-anesthetized mice. ERG waveforms obtained from mice anesthetized with a mixture of urethane, xylazine and ketamine or xylazine and ketamine were almost the same as those obtained from non-anesthetized mice. Therefore, the ERGs were recorded under mixed anesthesia, ketamine and xylazine, in the following study. Stable ERGs could be recorded after 40 min of dark-adaptation. 2. IAA at doses of 30 and 45 mg/kg caused slight depression of the amplitudes of the a-, b- and c-waves; however, these changes were no longer observed 7 days after dosing. At a dose of 60 mg/kg, the ERG waves were markedly depressed 1 day after dosing, and recovery was not observed until day 14. 3. Upon histopathologic examination of the retinas, a remarkable decrease in visual cells and thinning of the rod and cone layers and outer plexiform layer were observed with IAA at a dose of 60 mg/kg. 4. Using this newly developed recording technique, it was confirmed that stable ERGs could be recorded from pigmented mice repeatedly for 14 days, and the effects of IAA on the ERG could be detected. Histopathological abnormalities in the retinas correlated well with the changes in the ERGs. These results indicate that the newly developed ERG recording procedure is useful for evaluating retinal toxicity in pigmented mice.

[A procedure for recording electroretinogram (ERG) and effect of sodium iodate on ERG in mice].

A procedure for recording the electroretinogram (ERG) in mice with a coiled stainless steel-type electrode was developed in order to examine retinal toxicity. Mice received a single i.v. of sodium iodate (SI), a retinotoxic compound, via the tail vein at a dose of 12.5, 25, or 50 mg/kg, and the ERG was recorded periodically for 28 days after dosing. In addition, the retina was examined histopathologically on day 30 after dosing. 1. The mice were anesthetized with mixed anesthetics of urethane, xylazine and ketamine after 30 to 60 min of dark-adaptation. Sixteen responses to repetitive 1.2 J light stimuli at a frequency of 0.2 or 0.1 Hz were averaged by a microcomputer. Body temperature of the mice was kept constant at 37 to 38 degrees C using a thermostatically controlled heating mat. Under these conditions, stable ERG a-wave, b-wave, oscillatory potentials and c-wave could be recorded for 28 days. 2. SI at doses of 25 mg/kg or more caused depression of the amplitudes of the oscillatory potentials, and enhancement of the a- and b-wave amplitudes, while the c-wave was already extinguished on day 1 after dosing. Following these changes, the amplitudes of the a- and b-wave decreased from day 3 or 7 after dosing. These changes did not recover until day 28 after dosing. 3. Upon histopathologic examination of the retina, folding of the outer nuclear layer, disarrangement of the rods and cones, decrease of the visual cells and swelling and decrease of the pigment epithelial cells were observed with SI at 25 mg/kg or more. 4. Using this recording technique, it was confirmed that a stable ERG was recorded repeatedly for 28 days in mice, and the effects of SI on the ERG could be detected. Histopathologic findings in the retina revealed the abnormal portions were correlated well with the changes in the ERG. These results indicate that the ERG recording procedure developed in this study is useful for evaluating retinal toxicity in mice.

[Clinical observations on cefuzonam in pediatrics].

Cefuzonam (L-105, CZON) was given intravenously to 20 pediatric patients with the following acute bacterial infections: 13 of bronchopneumonia and 1 each of tonsillitis, purulent cervical lymphadenitis and acute tonsillitis, laryngitis, bronchitis, pyothorax, purulent meningitis complicated with septic arthritis, and urinary tract infection. Good clinical responses were obtained in all of the 20 patients and bacterial eradication of all 16 strains. No side effect was observed except 3 cases of slight elevation of transaminase, and 1 case each of soft stool and eosinophilia. From the above clinical results, it appears that CZON is a useful antibiotic for the treatment of pediatric patients with various kinds of bacterial infections.

[Defects of pyruvate metabolism in cultured lymphoblastoid cells of 20 patients with Leigh syndrome].

Lymphoblastoid cells are useful materials for the diagnosis and basic studies of many human genetic disorders. To elucidate the etiology of Leigh syndrome, biochemical analyses and mitochondrial DNA analyses were performed on cultured lymphoblastoid cells from 20 patients with the clinical characteristics of this disorder. In 9 of 20 cases, we were able to define the following defects. Eight patients had biochemical defects, including 3 with pyruvate dehydrogenase complex (PDHC), 3 with cytochrome c oxidase (complex IV), and 2 with NADH-cytochrome c reductase (complex I) deficiencies. Two of 3 patients with PDHC deficiency were diagnosed with thiamine-responsive PDHC deficiency. One patient had a point mutation (T-->G) of mitochondrial DNA at nucleotide position 8993. These results indicate that the underlying defects in Leigh syndrome are heterogeneous and cultured lymphoblastoid cells are very useful materials for diagnosis of the etiology of Leigh syndrome.